OPEN

Citation: Blood Cancer Journal (2016) 6, e441; doi:10.1038/bcj.2016.50

www.nature.com/bcj

REVIEW
‘Acute myeloid leukemia: a comprehensive review and
2016 update’
I De Kouchkovsky1 and M Abdul-Hay1,2

Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with an incidence of over 20 000 cases per year in the
United States alone. Large chromosomal translocations as well as mutations in the genes involved in hematopoietic proliferation
and differentiation result in the accumulation of poorly differentiated myeloid cells. AML is a highly heterogeneous disease;
although cases can be stratified into favorable, intermediate and adverse-risk groups based on their cytogenetic profile, prognosis
within these categories varies widely. The identification of recurrent genetic mutations, such as FLT3-ITD, NMP1 and CEBPA, has
helped refine individual prognosis and guide management. Despite advances in supportive care, the backbone of therapy remains
a combination of cytarabine- and anthracycline-based regimens with allogeneic stem cell transplantation for eligible candidates.
Elderly patients are often unable to tolerate such regimens, and carry a particularly poor prognosis. Here, we review the major
recent advances in the treatment of AML.

Blood Cancer Journal (2016) 6, e441; doi:10.1038/bcj.2016.50; published online 1 July 2016

INTRODUCTION absence of any large chromosomal abnormality.7 Studies of animal

Acute myeloid leukemia (AML) is the most common acute leukemia
in adults, accounting for ~ 80 percent of cases in this group.1 Within
the United States, the incidence of AML ranges from three to five
cases per 100 000 population. In 2015 alone, an estimated 20 830
new cases were diagnosed, and over 10 000 patients died from this
disease.2 The incidence of AML increases with age, from ~ 1.3 per
100 000 population in patients less than 65 years old, to 12.2 cases
per 100 000 population in those over 65 years. Although advances
in the treatment of AML have led to significant improvements in
outcomes for younger patients, prognosis in the elderly who
account for the majority of new cases remains poor.3 Even with
current treatments, as much as 70% of patients 65 years or older will
die of their disease within 1 year of diagnosis.4
PATHOPHYSIOLOGY
AML can arise in patients with an underlying hematological
disorder, or as a consequence of prior therapy (for example,
exposure to topoisomerases II, alkylating agents or radiation).5
However in majority of cases, it appears as a de novo malignancy
in previously healthy individuals. Regardless of its etiology,
the pathogenesis of AML involves the abnormal proliferation
and differentiation of a clonal population of myeloid stem cells.
Well-characterized chromosomal translocations, such as t(8:21) in
core-binding factor AML (CBF-AML) or t(15:17) in acute promye-
locytic leukemia (APL) result in the formation of chimeric proteins
(RUNX1-RUNX1T1 and PML-RARA, respectively), which alter the
normal maturation process of myeloid precursor cells. In addition
to large chromosomal rearrangements, molecular changes have
also been implicated in the development of AML. In fact, genetic
mutations are identified in more than 97% of cases,6 often in the
models at the turn of the century led to the development of
a two-hit model of leukemogenesis, which offers a conceptual
framework for classifying the various mutations associated with
AML.8 According to this model, class I mutations which result in the
activation of pro-proliferative pathways must occur in conjunction
with class II mutations which impair normal hematopoietic
differentiation in order for leukemia to develop.9,10 Common class
I mutations, such as FLT3 (internal tandem duplications, ITD, and
tyrosine kinase domain mutations, TKD), K/NRAS, TP53 and c-KIT are
found in ~ 28, 12, 8 and 4% of cases, respectively.7 Studies of solid
and hematological malignancies have also highlighted the role
of signal transducer and activator of transcription 3 (STAT3) in the
stimulation of cellular proliferation and survival.11–13 Enhanced
tyrosine phosphorylation of STAT3 whether due to increased
secretion of cytokines, such as IL-6(ref. 14) or mutations in receptor
tyrosine kinases (for example, FLT3 duplications15 or less frequently
JAK2)16 is seen in up to 50% of AML cases and signifies a worse
prognosis. Notable class II mutations include NPM1 and CEBPA,
which are found in ~ 27% and 6% of cases, respectively, and confer
a better prognosis.7 Alterations in genes involved in epigenetic
regulation have recently emerged as a third class of mutations, with
downstream effects on both cellular differentiation and prolifera-
tion. These include mutations in the DNA-methylation related genes
DNMT3A, TET2, and IDH-1 and IDH-2,6,7 which are found in more
than 40% of AML cases.
Despite significant advances, much remains to be discovered
on the exact contribution of these individual mutations
to the development of AML. As suggested by the ‘two-hit model,’
the pathogenesis and behavior of AML depends heavily on
the interactions between different somatic alterations and
chromosomal rearrangements. Thus, the c-KIT mutation has been

1
Department of Medicine, New York University School of Medicine, New York, NY, USA and 2Department of Hematology/Oncology, New York University Perlmutter Cancer
Center, New York, NY, USA. Correspondence: Dr M Abdul-Hay, Department of Hematology/Oncology, NYU School of Medicine, New York Perimutter Cancer Center, 240 East 38th
Street, 19 Floor, New York, NY 10016, USA.
E-mail: Maher.Abdulhay@nyumc.org
Received 13 April 2016; revised 3 May 2016; accepted 19 May 2016
 AML: a comprehensive review and 2016 update
I De Kouchkovsky and M Abdul-Hay
2
associated with t(8;21) or inv(16), and its presence carries significant
implications regarding prognosis. Similarly, NMP1 (a class II mutation)
frequently occurs in conjunction with the class I mutation FLT3-ITD,
or mutations in the epigenetic genes DNMT3A and IDH-1 or IDH-2.6
Most of the clinical manifestations of AML reflect the accumulation
of malignant, poorly differentiated myeloid cells within the bone
marrow, peripheral blood and infrequently in other organs. The
majority of patients presents with a combination of leuko-
cytosis and signs of bone marrow failure such as anemia and
thrombocytopenia. Fatigue, anorexia and weight loss are common
complaints; lymphadenopathy and organomegaly are not typically
seen. If left untreated, death usually ensues within months
of diagnosis secondary to infection or bleeding. The diagnosis
of acute leukemia is established by the presence of 20% or more
blasts in the bone marrow or peripheral blood.17 AML is further
diagnosed by demonstrating the myeloid origin of these cells
through testing for myeloperoxidase activity, immunophenotyp-
ing or documenting the presence of Auer rods. The latter finding
consists of azurophilic, often needle-shaped cytoplasmic inclusion
bodies that are commonly seen in APL, acute myelomonocytic
leukemia and the majority of AML with t(8;21). The diagnosis of
AML can also be established in the presence of an extramedullary
tissue infiltrate, or a documented t(8;21), inv(16) or t(15;17) in the
appropriate clinical setting, regardless of the blast percentage.18
CLASSIFICATION
The French–American–British classification system represents
the first attempt to distinguish between different types of AML.
Established in 1976, it defines eight subtypes (M0 through M7)
based on the morphological and cyto-chemical characteristics
of the leukemic cells. In 2001, as part of an effort to integrate
advances made in the diagnosis and management of AML, the
World Health Organization (WHO) introduced a new classification
system followed by a revised version in 2008.18 Later in 2016
a new revised version was released, the WHO classification of AML
distinguishes itself by incorporating genetic information with
Table 1. WHO classification of AML and related neoplasms
Types
AML with recurrent genetic abnormalities
AML with myelodysplasia-related changes
Therapy-related myeloid neoplasms
Myeloid sarcoma
Myeloid proliferations related to Down syndrome
Abbreviations: AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; ML, myeloid leukemia; WHO, World Health Organization.
Blood Cancer Journal
morphology, immunophenotype and clinical presentation to
define six major disease entities: AML with recurrent genetic
abnormalities; AML with myelodysplasia-related features; therapy-
related AML; AML not otherwise specified; myeloid sarcoma; and
myeloid proliferation related to Down syndrome (Table 1).19
Among cases of AML with recurrent genetic abnormalities, 11
subtypes are further delineated according to distinct chromoso-
mal translocations. In addition, the provisional entities AML with
mutated NPM1 and AML with mutated CEBPA were introduced
as part of the 2008 revision,18 while AML with BCR-ABL1 and
AML with mutated RUNX1 were introduced as part of the 2016
revision.19 Genetic abnormalities also inform the diagnosis of AML
with myelodysplasia-related changes: along with a history of MDS
or morphological evidence of dysplasia in two or more myeloid
cell lineages, the presence of myelodysplasia-related cytogenetic
abnormalities such as monosomy 5 or 7, and deletion 5q or 7q
identify cases of AML with myelodysplasia-related features.
PROGNOSTIC FACTORS
Accurate assessment of prognosis is central to the management
of AML. By stratifying patients according to their risk of treatment
resistance or treatment-related mortality (TRM), prognostic factors
help guide the physician in deciding between standard or
increased treatment intensity, consolidation chemotherapy or
allogenic hematopoietic stem cell transplant, or more fundamen-
tally in choosing between established or investigational therapies.
Among clinical factors, increased age and poor performance
status are both associated with lower rates of complete remission
(CR) and decreased overall survival (OS).3,20 Age and performance
status at diagnosis similarly help to predict the risk of TRM,
although multivariate model analyses suggest that other variables
such as platelet count, serum creatinine or albumin rather than
age itself account for most of the increased risk of TRM seen
in older patients.21 Therapy-related AML and AML associated with
a prior hematological malignancy also carry a significantly poorer
prognosis.22 Although clinical factors have an important role in
Genetic abnormalites
AML with t(8:21)(q22;q22); RUNX1-RUNX1T1
AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11
APL with PML-RARA
AML with t(9;11)(p21.3;q23.3); MLLT3-KMT2A
ML with t(6;9)(p23;q34.1); DEK-NUP214
AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2); GATA2, MECOM
AML (megakaryoblastic) with t(1;22)(p13.3;q13.3); RBM15-MKL1
AML with BCR-ABL1 (provisional entity)
AML with mutated NPM1
AML with biallelic mutations of CEBPA
AML with mutated RUNX1 (provisional entity)
AML with minimal differentiation
AML without maturation
AML with maturation
Acute myelomonocytic leukemia
Acute monoblastic/monocytic leukemia
Acute erythroid leukemia
Pure erythroid leukemia
Acute megakaryoblastic leukemia
Acute basophilic leukemia
Acute panmyelosis with myelofibrosis
Transient abnormal myelopoiesis
ML associated with Down syndrome

guiding therapy, cytogenetic changes constitute the single stron-
gest prognostic factor for CR and OS in AML. Accordingly, cases
of AML can be stratified into favorable, intermediate or adverse
prognostic risk groups based on their cytogenetic profile alone.
The chromosomal rearrangements t(8;21), t(15;17) or inv(16) all
confer a favorable prognosis,17,23 with a 3 year OS of 66% and 33%
in patients younger and older than 60 years, respectively.24 In
contrast, cytogenetic changes such as a complex karyotype (that is,
three or more chromosomal abnormalities in the absence of
any of the recurrent genetic abnormalities identified in the WHO
2008 classification), monosomy 5 or 7, t(6;9), inv(3) or 11q changes
other than t(9;11) have all been associated with a significantly
higher risk of treatment failure and death (Table 2).24 AML cases
with an intermediate prognostic risk mainly constitute of patients
with normal cytogenetics (CN-AML).17,24
Gene mutations have helped further refine risk stratification
based on cytogenetic changes alone. Among patients with t(8;21),
the presence of a c-KIT mutation significantly increases the risk
of relapse, and decreases OS to levels comparable to those of
patient with intermediate-risk AML.6,23,25 Although there is some
evidence that the presence of c-KIT mutations similarly lowers
prognosis in patients with inv(16),26 recent studies have failed
to show any prognostic impact in this subset of cases.6,27,28
Molecular changes have a particularly important role in refining
the prognosis of patients with CN-AML, which includes nearly
half of de novo AML cases. Thus CN-AML with a mutated CEBPA
or a mutated NPM1 in the absence of FLT3-ITD has been identified
as having a prognostic risk similar to that of AML with favorable
cytogenetic changes.17,29 The favorable prognostic impact
of CEBPA mutations has been further refined to biallelic mutations
only.30 On the other hand, multiple studies29,31 including a
meta-analysis of relapse-free survival (RFS) and OS in patients with
CN-AML o 60 years of age have consistently shown the presence
of FLT3-ITD to be associated with a worsened prognosis.6,32 This
has led to the classification of CN-AML with FLT3-ITD into
the adverse prognostic-risk group.23,33 As with CEBPA mutations,
the prognostic impact of FLT3-ITD may depend on the presence
of biallelic mutations. Several studies have shown a significantly
worse prognosis in patients with higher mutant to wild-type
allelic ratios.34,35 TP53 mutations, which are found in only 2– 8%
of cases,6,7 occur more frequently in cases with unfavorable
Table 2. Prognostic-risk group based on cytogenetic and molecular profile
Prognostic-risk group Cytogenetic profile alone
Favorable t(8:21)(q22;q22)
inv(16)(p13;1q22)
t(15;17)(q22;q12)
Intermediate CN-AML
t(9;11)(p22;q23)
Cytogenetic abnormalities not included in the
favorable or adverse prognostic risk groups
Adverse inv(3)(q21q26.2)
t(6;9)(p23;q34)
11q abnormalities other than t(9;11)
− 5 or del(5q)
−7
Complex karyotype
Abbreviations: AML, acute myeloid leukemia; ITD, internal tandem duplications.
AML: a comprehensive review and 2016 update
I De Kouchkovsky and M Abdul-Hay
3
cytogenetic and a complex karyotype.36 However regardless
of the cytogenetic profile, TP53 mutations are associated with
a very poor prognosis10 and may in fact represent the single worst
genetic prognostic factor.36 Mutations in DNA-related genes
also carry important implications for the prognosis and treatment
of AML. The presence of a mutated DNA methyl transferase
gene DNMT3A has been associated with a worsened prognosis
in CN-AML and adverse-risk AML.37 Partial tandem duplica-
tions of KMT2A (previously known as MLL), which encodes a
histone methyltransferase, have also been associated with a worse
prognosis in CN-AML.6,10,36 The prognostic impact of IDH-1/IDH-2
mutations is less well established and is likely modified by
co-occurring mutations. Among cases of FLT3-ITD-negative and
NPM1-mutated CN-AML, IDH-1/IDH-2 mutations have been
shown to improve OS.6 However a recent study of 826 patients
with known IDH-1 and IDH-2 status found no prognostic impact
on treatment response or OS.38 Further analysis is required
to delineate the role of DNA-related genes in OS and treatment
response. In addition to genetic profiling at the time of diagnosis,
information gained after treatment initiation plays a growing
role in refining patient prognosis: As could be expected,
individuals who achieve CR (defined morphologically as a blast
count of o5% of total nonerythroid cells in the bone marrow) after
induction therapy have a significantly increased survival compared
with patients with treatment resistant AML.39,40 Survival among
patients achieving CR is further influenced by the correction or
persistence of thrombocytopenia, with a shorter duration of survival
observed in the latter group.40 More recently, techniques such
as real-time PCR and flow cytometry have been used to measure
the presence of minimal residual disease among patients in CR.
Persistently elevated levels of RUNX1-RUNX1T1 transcripts after
induction therapy in patients with t(8;21) AML are thus associated
with an increased incidence of relapse.41,42 Similarly among patients
with intermediate-risk disease, detection of minimal residual disease
by flow cytometry is an independent predictor of relapse and
survival43,44 and carries important implications for management.45
ESTABLISHED TREATMENTS
Eligible patients first undergo induction therapy to achieve CR.
Unfortunately, minimal residual disease often persists in CR, and
Cytogenetic profile and molecular abnormalities
t(8:21)(q22;q22) with no c-KIT mutation
inv(16)(p13;1q22)
t(15;17)(q22;q12)
Mutated NPM1 without FLT3-ITD (CN-AML)
Mutated biallelic CEBPA (CN-AML)
t(8:21)(q22;q22) with mutated c-KIT
CN-AML other than those included in the
favorable or adverse prognostic group
t(9;11)(p22;q23)
Cytogenetic abnormalities not included in the favorable
or adverse prognostic risk groups
TP53 mutation, regardless of cytogenetic profile
CN with FLT3-ITD
CN with DNMT3A
CN with KMT2A-PTD
inv(3)(q21q26.2)
t(6;9)(p23;q34)
11q abnormalities other than t(9;11)
− 5 or del(5q)
−7
Complex karyotype
Blood Cancer Journal
 AML: a comprehensive review and 2016 update
I De Kouchkovsky and M Abdul-Hay
4
relapse will inevitably occur if treatment is discontinued. There-
fore, a favorable response to induction therapy should be followed
by consolidation therapy in order to eradicate any residual
disease and achieve lasting remission. The mainstay of induction
therapy consists of the ‘7+3’ regimen, which combines 7 days
of continuous infusion cytarabine with 3 days of anthracycline.
It is generally offered to patients with an intermediate to favorable
prognosis and a low risk of TRM (for example, younger patients
with good performance status, normal creatinine, albumin and
platelet count).23 Studies of induction regimens using either
daunorubicin at 60 or 90 mg/m2, or idarubicin at 12 mg/m2
have shown similar rates of CR and survival.23,46,47 A subset of
patients with DNMT3A and KMT2A mutations, which represents
a poor prognostic marker, may however benefit from higher
doses of daunorubicin.6 Standard dosing of cytarabine consists
of 100–200 mg/m2 daily administered as a continuous infusion
over 7 days. Although studies have shown greater efficacy at
higher doses, this added benefit is small and accrued at the cost
of increased toxicity;23,48,49 induction therapy with high-dose
cytarabine is generally reserved for refractory disease. The
combination of fludarabine, cytarabine, G-CSF and idarubicin
(FLAG-IDA), which was traditionally used for the treatment
of relapse, has also been shown to be a reasonable alternative
to standard induction regimens and results in similar CR rates and
OS overall but higher rates of CR after a single course.50
An optimal approach to elderly patients with AML has not
been established. Individuals over the age of 65 are more likely
to present with an adverse cytogenetic-risk profile, are less likely
to respond to chemotherapy and are often more susceptible to
treatment-related toxicities. However despite a significantly worse
prognosis, induction therapy improves survival in patients over
the age of 65 when compared with supportive care and palliative
chemotherapy,51 and should be pursued whenever possible.
Hypomethylating agents, traditionally used for the treatment
of myelodysplastic syndrome (MDS), have also shown efficacy
in elderly patients with AML. Evidence of a therapeutic benefit was
first demonstrated in post hoc analyses of patients with MDS who
were retrospectively found to meet diagnostic criteria for AML
under the WHO classification.52 A 2012 randomized trial of the
hypomethylating agent decitabine versus supportive care or low-
dose cytarabine in patients 65 years or older showed a significant
improvement in OS with hypomethylating therapy (although
this survival advantage failed to meet statistical significance in
the primary analysis).53 A more recent trial comparing the
hypomethylating agent azacitidine to supportive care, low-dose
cytarabine or standard induction therapy in patients 65 years or
older did not show any significant improvement in median OS.54
However among patients pre-selected to receive supportive
care, a subgroup analysis suggested a benefit to azacitidine
therapy. A similar benefit was seen among patients with adverse
cytogenetic-risk profile or MDR-AML.54 These results suggest
a promising role for the use of hypomethylating agents in older
individuals, including as a bridge for induction chemotherapy with
the goal of achieving CR.
Response to induction therapy should be evaluated 14 days
after initiation of treatment with a bone marrow aspirate and core
biopsy.17 Up to 25–50% of patients show persistent cytological
evidence of disease after one cycle of standard induction therapy
and require reinduction.55 Treatment options for such patients
(as well as patients with a disease relapse) include a second cycle
of standard dose cytarabine combined with an anthracycline,
high-dose cytarabine alone or FLAG-IDA, with roughly similar CR
rates of up to 50%.23,56,57 In addition, mitoxantrone-based regimens
(in combination with etoposide and/or cytarabine) have been shown
to achieve CR up to 40–60% of patients with recurrent or refractory
AML.58,59 Ultimately, around 60–80% of patients with de novo AML
will achieve CR with induction therapy.60 Patients in remission
should be offered consolidation therapy to eradicate residual disease
Blood Cancer Journal
and prevent relapse. Available options for consolidation include
chemotherapy and allogeneic hematopoietic stem cell transplant
(allo-HSCT). When choosing between these different options, the
risk of TRM should be weighted against the risk of treatment
failure or relapse. Intention-to-treat analyses (allocating patients
to allo-HSCT or chemotherapy based on the availability of a related-
donor) have found no benefit to allo-HSCT when compared with
chemotherapy in patients with cytogenetically favorable AML in first
CR.61,62 Thus chemotherapy is a reasonable first-line consolidation
choice for patients with a favorable prognosis. Regimens generally
consist of intermediate-dose cytarabine (two to four cycles each
consisting of six doses at 1.5–3 g/m2), which has been shown to be as
effective as high-dose cytarabine48,50,63 or multi-agent regimens.50
The optimal choice of consolidation therapy for patients with
an intermediate-risk cytogenetic profile but favorable genetic
mutations is more controversial: several studies have found
no benefit to transplantation in patients with NPM1-mutated,
FLT3-ITD-negative CN-AML.64,65 However in a recent intention-to-
treat analysis, allo-HSCT was shown to improve RFS in this
subset of patients.66 While these conflicting results may reflect
differences in study design, the difference in observed outcomes
may also reflect the modulating effect of co-occurring mutations,
such as IDH-1/-2.6 On the other hand allo-HSCT significantly
prolongs RFS and OS in some patients with intermediate-risk and
in most with adverse-risk AML, and should be offered as a first-line
consolidation therapy in eligible patients.62,67–69 In addition, allo-
HSCT has been shown to prolong RFS and improve OS in patients
with CN-AML and a high FLT3-ITD allelic ratio.70
NOVEL AGENTS
FLT3-ITD inhibitors
Inhibition of tyrosine kinase (TK) receptors has been used
successfully in various solid and hematological malignancies,
including Philadelphia-chromosome positive leukemias. Given the
prognostic impact and the high rate of FLT3 mutations, inhibition
of this TK has long been recognized as a potential therapeutic
target in AML. Tested agents include the first-generation inhibitors
sorafenib and midostaurin, as well as newer second-generation
agents such as quizartinib and crenolanib.
Sorafenib. Sorafenib is a tyrosine kinase inhibitors (TKI) of RAF
kinase, c-KIT, VGFR, PGFR and FLT3-ITD, which was first used
for the treatment of hepatocellular and renal cell carcinoma. As early
as 2008, phase I trials of sorafenib administered as a single agent
n patients with FLT3-ITD-positive relapsed or refractory (r/r) AML
demonstrated significant reductions in the number of leukemic cells
both in the peripheral blood and bone marrow, achieving CR in
several patients.71–74 In a phase II trial of 13 patients with r/r FLT3-
ITD-positive AML, single-agent sorafenib at doses of 200–400 mg
twice daily established CR (including CR with insufficient hemato-
logic recovery) in over 90% of cases. The agent was well-tolerated,
with grades 3 to 4 adverse events consisting of hyperbilirubinemia
(in 4/13 patients), elevated transaminases (5/13), diarrhea (4/13),
rash (2/13), pancreatitis (1/13), colitis (1/13), pericarditis (1/13),
hand and foot syndrome (2/13) and elevated creatinine (1/13).
Yet despite a strong initial response, the majority of patients
relapsed within 72 days of remission. Treatment failure was
associated with the emergence of D835Y and D835H mutations
within the FLT3 TKD. The addition of sorafenib to standard induction
regimens has yielded similarly mixed results: although initial phases I
and II trials of combination therapy were able to achieve longer
periods of disease-free survival, relapse invariably ensued within
several months of treatment.75–77 In one such study, the combina-
tion of sorafenib with cytarabine and idarubicin as induction and
consolidation therapy was able to achieve CR (or CR with an
incomplete platelet recovery) in 18 out of 18 patients with previously

untreated FLT3-ITD-positive AML.75 However after a median follow-up
of 9 months, more than half of these patients had relapsed.
Interestingly, no new mutation was observed in the FLT3 TKD of
the relapsed samples available for genetic sequencing. Alternative
mechanisms of resistance, such as the increased levels of FLT3
receptor ligand seen in patients receiving standard chemotherapy,
have been postulated to contribute to treatment failure.78 Combina-
tions of sorafenib and hypomethylating agents, which have not been
associated with an increase in FLT3 receptor ligand levels, are currently
being investigated. An encouraging phase II trial of sorafenib and
azacitidine in 43 patients with relapsed/refractory AML reported a
response rate of 46%, including 16% CR and 27% CR with incomplete
count recovery.79
The role of sorafenib as a first-line therapy in AML was further
delineated in two recent randomized trials: In 2013, Serve et al.
published the results of study, in which 201 patients 60 years or
older with newly diagnosed AML were randomized to receive
sorafenib or placebo in addition to standard chemotherapy.
Even within the subset of patients with FLT3-ITD-positive disease,
the addition of sorafenib did not result in an improved EFS or OS,
and was instead associated with an increased incidence of adverse
events.80 Most recently, Rollig et al.81 investigated the combina-
tion of sorafenib with standard chemotherapy in a multicenter
randomized controlled phase II trial of 267 patients age 60
or younger with newly diagnosed AML. In this study, patients
were randomly assigned to receive two cycles of standard ‘7+3’
induction therapy followed by three cycles of high-dose cytarabine
as consolidation therapy in combination with either sorafenib
(400 mg twice daily) or placebo. Patients assigned to the sorafenib
group also received 12 months of sorafenib maintenance therapy
after their last consolidation cycle. After 3 years the primary end
point, event-free survival, was achieved in 40% of patients in the
sorafenib group, versus 20% in the placebo group (with an
unadjusted hazard ratio of 0.64).81 The occurrence of grades 3–5
diarrhea, rash, fever and bleeding were significantly increased
among patients receiving sorafenib (occurring in 11, 7, 54 and 7%,
respectively). Importantly, only 17% of the individuals enrolled
in this study were positive for the FLT3-ITD mutation. The observed
benefit in FLT3-ITD-negative AML may in part be explained by off-
target inhibition of other tyrosine kinases, such as c-KIT, PGFR and
RAF kinase. Alternatively as suggested by the observed increased
in FLT3 receptor ligands level activation of wild-type FLT3 TK may
become a driver of leukemogenesis in patients receiving standard
chemotherapy.82 By targeting FLT3 TK in the period immediately
after cytarabine or daunorubicin therapy, sorafenib may exert
significant anti-leukemic activity even in FLT3-ITD-negative cells.
In an interesting parallel to the use of TKI in Philadelphia-
chromosome-positive leukemias, inhibition of FLT3 TK has also
shown a promising role in the post-allo-HSCT setting, either as
maintenance therapy or treatment of relapse. This was explored
recently by Chen et al.83 in a phase I trial of 22 patients with
FLT3-ITD-positive disease who received sorafenib as maintenance
therapy following allo-HSCT. In addition to establishing safety
and feasibility, the authors of this study reported rates of PFS and
OS which compared favorably to historical controls, particularly
in the subset of patients in CR1 or CR2 at the time of transplant.
In a retrospective analysis of six patients status post allo-HSCT,
sorafenib as maintenance therapy (n = 5) or treatment of relapse
(n = 1) resulted in a median PFS of 16 months, with all patients
remaining in molecular remission (that is, FLT3-ITD-negative
by PCR).84 Interestingly, skin graft-versus-host-disease occurred
shortly after initiation of therapy in five out of these six patients.
In addition to its action as a TKI, sorafenib may therefore possess
an immunomodulatory role, which synergizes with the graft-
versus-leukemia effect.85 Two phase I trials of sorafenib use in the
peri-transplant setting are currently underway (NCT01398501 and
NCT01578109).
AML: a comprehensive review and 2016 update
I De Kouchkovsky and M Abdul-Hay
5
Midostaurin. Midostaurin is another first-generation FLT3 TKI
with significant but transient single-agent activity in patients
with AML.86,87 As with sorafenib, its effects are limited by the
rapid emergence of resistance. Combinations of midostaurin
and existing chemotherapy regimens are currently under inves-
tigation: results of a phase I and combined phase I/II trial of
midostaurin and azacitidine have recently been published,
demonstrating the tolerability and efficacy of this combination in
patients with AML. In their cohort of 17 patients with a median age
of 73, Cooper et al.88 established a MTD level of 75 mg PO twice
daily with no observed dose-limiting toxicities. In another phase I/II
trial of the combination of midostaurin and azacitidine was tested
in patients with AML (primary or secondary) and MDS, Strati et al.
reported a lower MTD of 50 mg PO twice daily. The combination
achieved an overall response rate of 26%, with median remission
duration of 20 weeks. Although no DLT were observed in this
study, neutropenia, thrombocytopenia and anemia developed
in 96, 94 and 61% of patients, as well as infections and a decreased
left ventricular ejection fraction in 56 and 11% of patients,
respectively.89 In a recent randomized, double-blind trial of 717
patients with previously untreated FLT3 (ITD and TKD) positive AML,
Stone et al. explored the role of midostaurin in combination with
a standard ‘7+3’ induction regimen and high-dose cytarabine
consolidation therapy. Patients randomized to the midostaurin
treatment arm also received midostaurin as maintenance therapy
for one year. Although no difference in the rate of CR was observed
between the midostaurin and placebo arms, patients receiving
midostaurin had a significantly higher OS and EFS (with a hazard
ratio of 0.77 and 0.80, respectively).90
Quizartinib. Second-generation inhibitors such as quizartinib
have been designed to specifically target the FLT3 kinase, in order
to reduce toxicity from off-target effects. In addition to this
increased selectivity, quizartinib also possesses a good bioavail-
ability and a half-life of more than 24 h, which allows for a more
continuous FLT3 inhibition. A phase I study of oral quizartinib in
76 patients with relapsed/refractory AML was able to achieve
a hematological response in 30% of patients, and a CR in 13%
regardless of FLT3 mutational status. Among patients with FLT3-ITD,
the rate of hematologic response increased to 53%, with ~ 23%
of patients achieving CR. Patients were able to tolerate doses of
up to 200 mg/day, with grade 3 QT interval prolongation as the
only DLT.91 In one phase 2 trial, 137 patients with relapsed/
refractory AML were given quizartinib monotherapy. The agent was
administered in 28-day cycles at doses of 90 mg/day in females
and 135 mg/day in males; the most common treatment toxicities
were nausea and vomiting in 38 and 26% of patients, anemia in
29%, QT interval prolongation in 26%, febrile neutropenia in 25%,
diarrhea in 20% and fatigue in 20%. The rate of composite CR
(that is, CR, CR with incomplete platelet recovery and CR with
incomplete hematological recovery) and the median OS were 34%
and 25.6 months in FLT3-ITD-negative cases, and 44% and
23.1 months in FLT3-ITD positive cases. The observed benefit in
FLT3-ITD-negative disease may be explained by off-target effects,
or by the upregulation of the FLT3 TK pathway as suggested by the
rise in FLT3 receptor ligand levels in patients receiving standard
chemotherapy.92 Once again however, the response to FLT3 TKI is
limited by the rapid emergence of resistance: the median duration
of remission was only 5 weeks in patients with FLT3-ITD-positive
AML.93 Interim analysis of a phase I/II trial of quizartinib in
combination with azacitidine or cytarabine (NCT01892371) reported
an overall response rate of 82% in FLT3-ITD-positive AML, MDS or
chronic myelomonocytic leukemia.94 A phase III trial comparing
quizartinib monotherapy to salvage chemotherapy in relapsed and
refractory AML (NCT02039726) is similarly underway.
Crenolanib. Crenolanib besylate is an orally available second-
generation FLT3 TKI, with activity against FLT3-ITD and FLT3-TKD
Blood Cancer Journal
 AML: a comprehensive review and 2016 update
I De Kouchkovsky and M Abdul-Hay
6
mutants. Unlike other FLT3 TKIs, which are subject to the
emergence of resistance conferring kinase domain mutations
(such as D835Y), crenolanib appears to possess extensive ‘pan-
kinase’ inhibition of secondary TKD mutations. Using concentra-
tions far below the clinically achievable plasma levels, Smith et al.
were unable to identify any single TKD mutation able to confer
resistance to crenolanib.95 In a phase II study of 38 patients with
FLT3-mutated AML (including relapsed and refractory patients),
crenolanib administered at doses of 200 mg/m2 per day three
times a day in 28 days cycle achieved a median EFS and OS of
8 and 19 weeks, respectively.96 Crenolanib is currently being
studied in multiple clinical trials in AML patients, both with and
without FLT3-mutated AMLs.
STAT inhibitors
STAT3 tyrosine phosphorylation is upregulated in up to 50% of AML
cases and confers a worse prognosis. Activation of the STAT3
signaling pathway is also stimulated by the FLT3 receptor ligand,97
and may represent a key step in the development of FLT3 TKI
resistance. Several small molecules of STAT3 inhibitors have been
developed and are currently being investigated for the treatment
of AML: in 2011, Redell et al. showed decreased STAT3 phosphory-
lation and induction of apoptosis in AML cell lines treated with the
STAT3 inhibitor C188-9.98 More recently, the optimized compound
MM-206 demonstrated a dose-dependent induction of apoptosis
in AML cell lines cultured in the presence of bone marrow stromal
cells. The anti-tumor activity of MM-206 was confirmed in vivo
by reducing blast count and improving survival in AML-engrafted
mice.99 OPB-31121 is a small molecule inhibitor of STAT3 and STAT5
phosphorylation, which has demonstrated activity in advanced
solid tumors.100 Treatment of various leukemic cell lines with this
compound resulted in significant growth inhibition, including
FLT3-ITD-positive AML cells.101 Importantly, OPB-31121 was able
to overcome FLT3 receptor ligand-induced STAT3 phosphoryla-
tion, and may help to prevent the emergence of resistance in
patients receiving FLT3-ITD TKI. Other STAT3 inhibitors that work
as antisense oligonucleotide (ASO) for STAT3 are in clinical trials in
hematological malignancies including AML and will have to wait
to see their efficacies in near future.
IDH1/IDH2 small molecule inhibitors
Gain of function mutations in IDH-1 and IDH-2 enzymes are found
in approximately 20% of cases.7 Recent attempts have been
made to target these mutant enzymes as a potential treatment
for AML. In 2013, Wang et al.102 published the results of AGI-6780,
a small molecule inhibitor of the R140Q mutant IDH-2 enzyme. In an
ex vivo model of primary human AML cells, treatment with AGI-6780
was able to overcome the differentiation block of leukemic cells.
Recently, the IDH-2 inhibitor AG-221 was found to confer a dose-
dependent survival benefits in a primary human IDH-2 mutant
AML xenograft model. At a cellular level, AG-221 treatment was
associated with an initial phase of CD45+ blast cells proliferation,
followed by cellular differentiation.103 A phase I trial of AG-221
in IDH-2 mutant leukemia is currently underway (NCT01915498).
IDH-1 mutant enzymes are also the targets of new therapeutic
inhibitors: Preliminary results of a phase I trial of the small molecule
inhibitor AG-120 (NCT02074839) demonstrated hematological
response in 7 out of 14 IDH-1 positive patients, including 4 CR.104
Clofarabine
Clofarabine is a second-generation purine nucleoside analog
approved for the treatment of relapsed or refractory pediatric
acute lymphocytic leukemia. In AML, it has shown activity and
tolerability as a single-agent, administered intravenously at doses of
20–30 mg/m2 for 5 days, with overall response rates of ~ 40%.105,106
In a 2008 randomized study of 70 patients aged 60 years and older,
Blood Cancer Journal
it was reported a CR rate of 63% using the combination of
clofarabine with low-dose cytarabine, compared with 31% with
clofarabine alone.107 In a study of 320 patients over the age of 55
with relapsed/refractory AML, the combination of clofarabine and
cytarabine achieved significantly higher rates of CR, CR with
incomplete platelet count and DFS when compared with cytarabine
alone.108 Although neither of these studies was able to show
an improved OS, these results suggest a synergistic action between
clofarabine and cytarabine, and have spurred interest in the
combination of these two agents. Recently a study was published
showing the results of a phase 2 trial of clofarabine and low-dose
cytarabine in older patients with newly diagnosed AML.109 In this
study, 118 patients age 60 or older (median age of 68 years)
received induction therapy with clofarabine at 20 mg/m2 on day 1
through 5 and low-dose cytarabine at 20 mg subcutaneously
twice daily on day 1 through 10. In an attempt to improve survival,
this was followed by consolidation and maintenance therapy with
up to 18 cycles of clofarabine and low-dose cytarabine alternating
with decitabine. CR was achieved in 60% of cases, with a median
OS of 11.1 months, and a median RFS of 14.1 months. The regimen
was well-tolerated, with a 4-week mortality rate of 3%. The most
common non-hematological toxicities reported in this study
included nausea in 81% of cases, elevated liver transaminases
and bilirubin in 64 and 47% of cases, as well as rash in 56% of
cases. Clofarabine may thus represent a well-tolerated addition to
low-dose cytarabine in elderly patients unable to receive standard
chemotherapy or allo-HSCT for consolidation therapy. Clofarabine
has shown similarly promising results in younger patients when
combined with the standard ‘7+3’ induction regimen. In a study of
57 newly diagnosed patients under the age of 60, the combination
of clofarabine (at 22.5 mg/m2 IV daily for 5 days) with idarubicin
and cytarabine used as a frontline induction and consolidation
therapy achieved CR rates of 74% and a median EFS of 13.5 months
(the median OS and RFS had not been reached by a median follow-
up of 10.9 months).110 A phase I/II study of frontline clofarabine,
cytarabine and idarubicin in patients with intermediate and poor
risk AML is currently underway (NCT00838240). The potential role
of clofarabine in the peri-transplant setting is also currently under
investigation. In a multicenter two-stage phase II trial, 84 patients
with relapsed and refractory AML received clofarabine (at 30 mg/m2
for 5 days) in combination with cytarabine as salvage therapy,
followed by 4 days of clofarabine and one dose of melphalan
in chemo-responsive patients with HLA-compatible donors. Out of
the 56 patients who underwent allo-HSCT, CR was achieved in 50
(including 11 CR with incomplete platelet recovery and 10 CR by
chimerism). The 2-year OS of 43% compared favorably with historical
controls.111 Although randomized controlled trials are needed to
clearly delineate the role of clofarabine in AML these studies present
promising results in support of this agent, particularly in combina-
tion with cytarabine in the elderly.
In addition to its activity as an intravenous agent, clofarabine
has also captured interest as an oral agent for the treatment of AML.
Unlike previous purine nucleoside analogs, clofarabine is able to
resist acidic pH as well as phosphorolytic cleavage by gastro-
intestinal Escherichia coli. As a result it possesses a bioavailability of
roughly 50%.112 In a phase I/II study of 35 patients 60 years or older
with relapsed/refractory AML or high-risk MDS the feasibility and
efficacy of oral clofarabine combined with low-dose cytarabine was
investigated as a first-line therapy.113 At a MTD of oral clofarabine
of 20 mg per day for 5 days, CR was achieved in 42% of patients
(including CR with incomplete count recovery in 4%). Most
importantly, more than 50% of cycles administered at the MTD
were given in the outpatient setting. In a population at an increased
risk of TRM, oral clofarabine may thus offer a valuable addition to
reduced intensity chemotherapy.

Monoclonal antibodies
Monoclonal antibodies exert their anti-tumor activity through
direct antibody-dependent cytotoxicity or through the conjuga-
tion of cytotoxic agents, which allows for the targeted delivery
of potent chemotherapy to neoplastic cells. Gemtuzumab
ozogamicin (GO) is a humanized recombinant antibody directed
at CD33, a transmembrane protein expressed on cells of myeloid
lineage. The antibody is conjugated to the DNA-cleaving cytotoxic
agent calicheamicin, and is internalized by CD33-positive cells. GO
received FDA approval in 2000 for the treatment of CD33-positive
AML in patients 60 years or older at first relapse. However in 2009,
interim analysis of a randomized clinical trial of 637 patients
with newly diagnosed AML revealed increased fatal toxicity with
no improvement in CR, disease-free survival or OS in patients
receiving GO in combination with standard chemotherapy.114 The
trial was prematurely terminated and FDA approval was rescinded.
Despite its removal from the market, a 2014 meta-analysis of five
randomized clinical trials demonstrated a decrease in relapse
and improved survival in patients receiving GO in addition to
standard chemotherapy.115 A subset analysis further revealed
that this survival benefit was limited to patients with a favorable or
intermediate cytogenetic-risk profile. Most recently, a randomized
trial of 237 patients 60 years of age or older ineligible for intensive
chemotherapy showed an improved OS (hazard ratio of 0.69) in
those assigned to GO induction and consolidation compared
with best supportive care.116 This survival benefit was again
most pronounced in patients with an intermediate to favorable
cytogenetic-risk profile. Although further studies are required
to fully delineate the effects of gemtuzumab in the treatment
of AML, these data suggest a benefit among elderly patients
with favorable or intermediate cytogenetic-risk profile. CD37 is
a transmembrane protein that is expressed in high levels on
maturing B cells. Although its exact function has not yet been
elucidated, it is upregulated in non-Hogkin lymphoma and chronic
lymphocytic leukemia.117 Initially conceived as a therapy for B-cell
malignancies, AGS67E is a fully human anti-CD37 IgG antibody
that is conjugated with monomethyl auristatin E (MMAE), a potent
microtubule-disrupting agent. AGS67E allows for the selective
delivery of MMAE to CD37-positive malignant cells and results
in apoptosis. Although CD37 is minimally expressed on normal
myeloid stem cells it was recently demonstrated to have differential
expression of CD37 on the surface of CD34+/CD38− AML stem cells.
In vitro treatment of leukemic cells with nanomolar concentrations
of AGS67E resulted in cytotoxicity, altered cell growth and apoptosis
in seven out of 16 AML cell lines. The administration of AGS67E was
further found to significantly decreased tumor engraftment in
a murine xenograft model of AML, resulting in undetectable
leukemic cell levels in three out of four AML samples. CD37 may
thus represent a novel therapeutic target for the selective inhibition
of leukemic cell growth. Although still at an early stage of its clinical
development, AGS67E is a promising new therapy. More impor-
tantly, the identification of unique molecular markers expressed on
the surface of leukemic cells is an emerging avenue for the
discovery of novel targeted therapies against AML.
CART therapy
Chimeric antigen receptors are synthetic T-cell receptors with
antibody-like specificity. They combine a single-chain variable
fragment from a monoclonal antibody with the transmembrane
and intracellular domains of a T-cell receptor. This allows for the
creation of a host-derived population of chimeric antigen receptor-T
(CART) cells, which can be directed at a pre-determined antigen.
CD19-directed CART cell therapy has shown exciting efficacy in the
treatment of acute lymphoblastic leukemia and B-cell lymphoma.
Although this treatment does not distinguish between malignant
and healthy CD19 cells, patients tolerate the depletion of CD19
lymphocytes with relatively little morbidity. In contrast to this,
AML: a comprehensive review and 2016 update
I De Kouchkovsky and M Abdul-Hay
7
depletion of the normal cells of myeloid lineage is associated
with unacceptable neutropenia, and attempts to apply CART cell
therapy to the treatment of AML have had to focus on identifying
an appropriate antigen to target malignant cells while sparing
non-malignant myeloid cells. Kenderian et al. recently developed
a CD33-specific CAR based on the single-chain variable fragment
of gemtuzumab ozogamicin. They have reported potent in vitro
activity of such CD33-specific CART cells against AML cell lines.118
CD33-specific CART therapy also prolonged survival in AML
xenografts. However CD33 is expressed on normal cells of myeloid
lineage and the reported anti-tumor effects were associated
with profound cytopenias. Using electroporation of CD33-specific
CAR mRNA into human T cells, the authors are able to induce the
transient expression of anti-CD33 CAR,118 which may confer
clinically significant anti-tumor activity while avoiding long-term
myelosuppression. In an attempt to selectively target leukemic
myeloid cells, others have focused instead on the β member of the
folate receptor family (FRβ). This receptor subtype is primarily
expressed on myeloid-lineage hematopoietic cells, and is upregu-
lated in the setting of malignancy.119 FRβ is expressed in 70% of
cases of primary AML, and its expression can be further upregulated
following treatment with all-trans retinoic acid.120 In a recent
publication the effect of FRβ-specific CART cells therapy in vitro as
well as in AML xenograft was reported. The study demonstrated lytic
activity against FRβ positive AML cell lines both in vitro and in vivo.
Importantly, no evidence of toxic activity against healthy human
CD34-positive stem cells was observed in vitro.121 Although still in its
early stages, CART cell therapy may thus provide an alternative
mechanism of treatment for patients with relapsed/refractory AML.
CONCLUSION
AML is a biologically and clinically heterogeneous disease. Although
advances in supportive care and prognostic risk stratification
have optimized established therapies, overall long-term survival
remains poor. Elderly patients who account for the majority of
newly diagnosed cases are more likely to present with an adverse
cytogenetic profile. At the same time the increased risk of TRM often
precludes this population of patients from receiving optimal
chemotherapy or stem cell transplantation. Novel targeted therapies
offer the promise of effective anti-leukemic activity with reduced
toxicity from off-target effects. However given the molecular
diversity of AML, it is unlikely that targeted therapies such as FLT3
tyrosine kinase inhibitors will provide a single ‘magic bullet’ against
this disease. Rather the development of new treatments, in concert
with improved genetic profiling and risk stratification, can be
expected to result in incremental gains in remission and survival.
Furthermore in addition to mutated enzymes and upregulated
pathways, the identification of unique cell surface markers can
provide a therapeutic target for recombinant monoclonal antibodies
or chimeric antigen receptors. Here, the challenge lays in selectively
targeting leukemic myeloid cells while sparing non-malignant
myeloid precursors. Lastly, the development of well-tolerated oral
therapies, such as clofarabine, will increasingly broaden the range of
available treatment for elderly patients at a higher risk of mortality
from standard chemotherapy regimens. We are looking to a new era
in the treatment of AML to begin with novel agents so we can
achieve better responses with prolong OS particularly for patients
with relapsed or refractory diseases and poor cytogenetic features.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Blood Cancer Journal
 AML: a comprehensive review and 2016 update
I De Kouchkovsky and M Abdul-Hay
8
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